Importance of the 5’ untranslated region for recombinant enzyme production in isolated Bacillus subtilis 007

The production of industrial enzymes requires an efficient expression system with a suitable host. This study investigated the isolated Bacillus subtilis 007 as a host for expressing three enzymes with potential application in the food industry. Firstly, testing the P_aprE and P₄₃ promoters and the corresponding 5' untranslated regions revealed great differences in the production of the recently discovered β-galactosidase from Paenibacillus wnnyii. Expression controlled by the P_aprE promoter yielded a significantly higher activity of 2515 µkat/L, compared to 56 µkat/L with the P₄₃ promoter. Modifications on the P_aprE core promoter region or the spacer, the sequence between the Shine-Dalgarno sequence and the start codon, did not improve β-galactosidase production. Since the aprE 5' untranslated region contributes to a high mRNA stability, it was incorporated into the P₄₃ construct to determine whether mRNA stability is responsible for the differences observed in β-galactosidase production. Interestingly, mRNA stability was significantly improved and led to a nearly 50-fold higher β-galactosidase production of 2756 µkat/L. This strategy was successfully validated by the expression of two other enzymes: the cellobiose-2-epimerase from Caldicellulosiruptor saccharolyticus and the β-glucosidase from Pyrococcus furiosus. These findings underscored the crucial role of post-transcriptional regulation and emphasized mRNA stability as a key role in recombinant enzyme production in B. subtilis 007.